Medical Research Center, The First Affiliated Hospital of Wenzhou Medical University, 1 Xuefubei Street, Ouhai District, Wenzhou, 325000, Zhejiang Province, China.
Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, 1 Xuefubei Street, Ouhai District, Wenzhou, Zhejiang Province, China.
Clin Epigenetics. 2024 Mar 26;16(1):48. doi: 10.1186/s13148-024-01658-2.
miR-182 promoter hypermethylation frequently occurs in various tumors, including acute myeloid leukemia, and leads to low expression of miR-182. However, whether adult acute lymphocyte leukemia (ALL) cells have high miR-182 promoter methylation has not been determined.
To assess the methylation status of the miR-182 promoter, methylation and unmethylation-specific PCR analysis, bisulfite-sequencing analysis, and MethylTarget™ assays were performed to measure the frequency of methylation at the miR-182 promoter. Bone marrow cells were isolated from miR-182 knockout (182KO) and 182 wild type (182WT) mice to construct BCR-ABL (P190) and Notch-induced murine B-ALL and T-ALL models, respectively. Primary ALL samples were performed to investigate synergistic effects of the hypomethylation agents (HMAs) and the BCL2 inhibitor venetoclax (Ven) in vitro.
miR-182 (miR-182-5P) expression was substantially lower in ALL blasts than in normal controls (NCs) because of DNA hypermethylation at the miR-182 promoter in ALL blasts but not in normal controls (NCs). Knockout of miR-182 (182KO) markedly accelerated ALL development, facilitated the infiltration, and shortened the OS in a BCR-ABL (P190)-induced murine B-ALL model. Furthermore, the 182KO ALL cell population was enriched with more leukemia-initiating cells (CD43B220 cells, LICs) and presented higher leukemogenic activity than the 182WT ALL population. Furthermore, depletion of miR-182 reduced the OS in a Notch-induced murine T-ALL model, suggesting that miR-182 knockout accelerates ALL development. Mechanistically, overexpression of miR-182 inhibited proliferation and induced apoptosis by directly targeting PBX3 and BCL2, two well-known oncogenes, that are key targets of miR-182. Most importantly, DAC in combination with Ven had synergistic effects on ALL cells with miR-182 promoter hypermethylation, but not on ALL cells with miR-182 promoter hypomethylation.
Collectively, we identified miR-182 as a tumor suppressor gene in ALL cells and low expression of miR-182 because of hypermethylation facilitates the malignant phenotype of ALL cells. DAC + Ven cotreatment might has been applied in the clinical try for ALL patients with miR-182 promoter hypermethylation. Furthermore, the methylation frequency at the miR-182 promoter should be a potential biomarker for DAC + Ven treatment in ALL patients.
miR-182 启动子的高甲基化经常发生在各种肿瘤中，包括急性髓系白血病，并导致 miR-182 的低表达。然而，成人急性淋巴细胞白血病（ALL）细胞是否具有高 miR-182 启动子甲基化尚未确定。
为了评估 miR-182 启动子的甲基化状态，进行了甲基化和非甲基化特异性 PCR 分析、亚硫酸氢盐测序分析和 MethylTargetTM 测定，以测量 miR-182 启动子的甲基化频率。从 miR-182 敲除（182KO）和 182 野生型（182WT）小鼠的骨髓细胞中构建 BCR-ABL（P190）和 Notch 诱导的鼠 B-ALL 和 T-ALL 模型，分别。对原发性 ALL 样本进行了研究，以研究低甲基化剂（HMAs）和 BCL2 抑制剂 venetoclax（Ven）在体外的协同作用。
由于 ALL blasts 中的 miR-182 启动子 DNA 高甲基化，miR-182 的表达在 ALL blasts 中明显低于正常对照（NCs），而在正常对照（NCs）中则没有。miR-182 敲除（182KO）显著加速了 ALL 的发展，促进了浸润，并缩短了 BCR-ABL（P190）诱导的鼠 B-ALL 模型中的 OS。此外，182KO ALL 细胞群富含更多的白血病起始细胞（CD43B220 细胞，LICs），并且比 182WT ALL 细胞群具有更高的白血病发生活性。此外，miR-182 的耗竭降低了 Notch 诱导的鼠 T-ALL 模型中的 OS，表明 miR-182 敲除加速了 ALL 的发展。机制上，miR-182 的过表达通过直接靶向 PBX3 和 BCL2 抑制增殖并诱导细胞凋亡，PBX3 和 BCL2 是两个众所周知的癌基因，是 miR-182 的关键靶点。最重要的是，DAC 联合 Ven 对 miR-182 启动子高甲基化的 ALL 细胞具有协同作用，但对 miR-182 启动子低甲基化的 ALL 细胞没有协同作用。
总的来说，我们确定 miR-182 是 ALL 细胞中的肿瘤抑制基因，由于高甲基化导致 miR-182 的低表达促进了 ALL 细胞的恶性表型。DAC+Ven 联合治疗可能已应用于 miR-182 启动子高甲基化的 ALL 患者的临床尝试中。此外，miR-182 启动子的甲基化频率可能是 ALL 患者 DAC+Ven 治疗的潜在生物标志物。
